Project Summary The deadliest form of human malaria is caused by the eukaryotic parasite Plasmodium falciparum, which is responsible for nearly 450,000 deaths every year. Nearly half of the world?s population lives in areas where malaria is endemic, resulting in almost ~250 million infections each year. As yet, there are no effective vaccines against malaria and antimalarial drugs are the mainstay of treatment. Unfortunately, the parasite has gained resistance to all antimalarial drugs used in the clinic and these drug-resistant strains are spreading throughout the world. Thus, it is crucial that we constantly identify potential novel drug targets to stay ahead of this deadly disease. Understanding the signaling pathways that drive the biology of the parasite will provide new antimalarial drug targets that are unique to the parasite and absent in the host. Calcium ion (Ca2+) signaling has emerged as one of the major drivers of the life cycle of P. falciparum. The goal of this proposal is to study Ca2+ signaling and the pathways that regulate ion fluctuations in malaria parasites. In P. falciparum, similar to other eukaryotic organisms, the cytoplasmic levels of Ca2+ is very low and its concentration rises in the cytoplasm in response to specific signals. This increased cytosolic Ca2+ results in a signaling cascade that that is essential for the life cycle of the parasite. Once the signal subsides, the levels of cytosolic Ca2+ falls back via uptake into intracellular Ca2+ stores, such as the endoplasmic reticulum. The P. falciparum genome lacks several canonical genes that are known to be essential for Ca2+ signaling in other well- studied eukaryotic organisms. Therefore, we will target the only soluble protein with Ca2+ binding domains that localizes to the major intracellular Ca2+ store, the endoplasmic reticulum. We hypothesize that this protein regulates the release and uptake of Ca2+ from this organelle. Our preliminary data show that this gene is essential for the asexual life cycle of the parasite and is required for the invasion of the parasite into its host red blood cell. We will utilize genetic, cellular, and biochemical approaches to define the role of this gene in regulating Ca2+ signaling and the invasion of P. falciparum into the host cell. These include the use of genetically encoded Ca2+ indicators to reveal the fluctuations of Ca2+ during the intraerythrocytic life cycle of P. falciparum as well as the effect of genetic interventions on the homeostasis of Ca2+. A second independent proximity-based labeling approach will be undertaken to isolate and discover novel partners of the targeted gene to define the network of genes required to regulate the flow of Ca2+ within the P. falciparum infected human red blood cells. Achieving the aims of this study will reveal the essential parasite-specific pathways that regulate Ca2+ signaling, which can be targeted for antimalarial drug development.